Energy, Ecology and Environment

, Volume 3, Issue 2, pp 87–94 | Cite as

Soda lignin from Citrus sinensis bagasse: extraction, NMR characterization and application in bio-based synthesis of silver nanoparticles

  • Caio H. N. Barros
  • Danijela Stanisic
  • Bruna F. Morais
  • Ljubica Tasic
Original Research Article


Lignin is one of the most abundant natural materials with many important roles, especially in providing structural resilience of plants. It is formed through the radical polymerization of aromatic monomers and shows structural and compositional differences depending on sources, biosynthesis and processes used for its extraction. Herein, we present extraction of lignin from the Citrus sinensis (sweet orange) bagasse using full sequential extraction in a yield of 0.34% and report on the soda lignin nuclear magnetic resonance (NMR) properties (1H NMR and 2D NMR). The soda lignin was then applied in the sustainable synthesis of silver nanoparticles (AgNPs). The obtained silver nanoparticles showed unimodal distribution of sizes, spherical morphology, average diameters of 19.1 ± 4.7 nm and negative zeta potentials of − 28.5 ± 3.2 mV. The AgNPs were also found to be stable over several months.


Citrus sinensis (sweet orange, C. sinensis) bagasse Lignin Silver nanoparticles 

1 Introduction

Lignin is an aromatic material that is biosynthesized through polymerization of 4-hydroxycinnamyl (p-coumaryl alcohol), coniferyl and sinapyl alcohols (Fig. 1), which are phenylalanine-derived monomers (Mansfield et al. 2012; Zeng et al. 2013; Constant et al. 2016; Dewick 2002). The relative amount of each monomer, monolignol, can vary according to the plant type. The alcohols are mainly synthesized by reduction of cinnamic acid; however, their substituents are not necessarily introduced during this phase, as esters and aldehydes may also act as substrates for aromatic hydroxylation and methylation (Dewick 2002).
Fig. 1

Biosynthetic pathway of 4-hydroxycinnamyl (p-coumaryl alcohol), coniferyl and sinapyl alcohols.

Adapted from Dewick (2002)

Coniferyl, p-coumaryl and sinapyl alcohol monomers can couple to form linkages as exemplified by guaiacyl glycerol β-coniferyl ether (β-arylether linkage), dehydrodiconiferyl alcohol (phenylcoumaran linkage) and pinoresinol (resinol linkage). These dimers can further react by similar mechanisms to produce a lignin material that contains a heterogeneous series of intermolecular bonds. In contrast to other natural materials, lignin shows ordered repeating units with around 50–70% of the β-aryl ether-type linkages. Dimeric units are also found in nature and are called lignans (Dewick 2002; Constant et al. 2016). The monolignols, such as coniferyl, p-coumaryl and sinapyl alcohols, when inserted in the biopolymer structure, have their names changed to guaiacyl, p-hydroxyphenyl and syringyl residues (Laurichesse and Avérous 2014).

Although lignin is one of the most prevalent polymers in nature, its composition and concentration in plant material can vary. The specificity of the structure, as well as the type of plant from which it is isolated, influences largely on lignin molar mass distributions. As Constant et al. (2016) determined, molar masses of six technical lignins varied from 100 to 3,000,000 g mol−1. The process and type of isolation may also have significant influences on the lignin final structure. Residual carbohydrates, such as xylan, arabinan, galactan, glucan and mannan, are found to be covalently bonded to lignin compounds, and their content can vary from 0.2 to 2.4% (Constant et al. 2016). Few studies have been published on lignin extracted from oranges. Ververis et al. (2007) reported that lignin accounts for 2.1 ± 0.3% of the orange peel organic material. Aravantinos-Zafiris et al. (1994) analyzed the fiber fraction from orange peels after pectin extraction and determined that 0.6% of this material accounted to lignin. However, reports on different types and composition of the orange extracted lignin are scarce.

On the other hand, silver nanoparticles (AgNPs) as one of the most studied nanomaterials are used for numerous applications, principally due to their well-known antimicrobial activities (Rai et al. 2009). Their size in the nanometer range leads to a high surface/area ratio, which contributes to an elevated toxicity toward bacteria through the combination of various types of mechanisms of action (Durán et al. 2016a, b). Moreover, AgNPs applications, such as nanosensors (Yola et al. 2014), drug delivery agents (Adeyemi and Sulaiman 2015), gene therapy agents (Marin et al. 2015), antifungal (Ballottin et al. 2017) and anti-tick (Durán et al. 2017) nanomaterials, were investigated. The synthetic route for AgNP synthesis has also been a subject of intense study. While classical chemical methods are becoming less frequent, new green and/or bio-based syntheses are emerging as good alternatives (Sharma et al. 2009) for AgNP synthesis. The use of fungi (Ballottin et al. 2016), plant extracts (Ahmed et al. 2016) and bacteria (Das et al. 2014) for the biosyntheses of AgNPs are commonly seen in scientific reports, and nowadays, several commercial products contain nanoparticles of these types.

Along with the urge of using sustainable, non-toxic materials for the production of silver nanoparticles, the utilization of renewable biomaterials has been intensified in the last decades. These materials must possess some of the following characteristics: biodegradability, recyclability, ease of separation, no health risks, and others (Thakur et al. 2014), as to be considered renewable. Plant biomasses are great sources of sustainable, renewable materials, such as cellulose, hemicelluloses and lignin. All of these are great candidates for applications in fuels, polymers and green materials (Duval and Lawoko 2014; Constant et al. 2016; Watkins et al. 2015; Azadi et al. 2013).

Lignin, due to the existence of the aforementioned phenol groups, is a good candidate for silver ions reduction and, therefore, for silver nanoparticles production. In fact, green synthesis of silver nanoparticles from lignin has been reported using lignin as reducing and stabilizing agent (Hu and Hsieh 2015). Hu and Hsieh (2015) were able to produce cellulose-bound antibacterial AgNPs through the reduction of silver ions by lignin and observed that longer synthesis times and cellulose fibers influenced on the polydispersion of the nanoparticles (5–100 nm). The same research group then reported a study on the kinetics and yield of AgNPs syntheses using alkali (low sulfonate) lignin, where spherical nanoparticles in the size range between 4 and 20 nm (Hu and Hsieh 2016) were obtained. Similarly, Shen et al. (2014) reported on the sophisticated synthesis of AgNPs from lignin in a Tollens’ reaction, which resulted in spherical nanoparticles with a mean size of 24 nm with interesting heavy metal detection properties.

In this paper, we report on the sequential extraction of soda lignin from the orange (C. sinensis) bagasse for the first time. Orange bagasse lignin was characterized, mainly by nuclear magnetic resonance (NMR), and used for the production of highly stable silver nanoparticles. Thus, this renewable green material extracted from the orange waste—soda lignin—can be very successfully used for bio-nanotechnology purposes.

2 Experimental

2.1 Extraction of soda lignin from orange bagasse

Industrial orange bagasse was submitted to a sequential extraction of essential oils, hesperidin, sugars and pectin prior to the actual lignin extraction as to obtain pure soda lignin. One hundred grams of dry orange bagasse was submitted to extraction of essential oils with petroleum ether (500 mL) for 1.5 h in a Soxhlet extractor. The solvent was then changed to methanol (500 mL) to extract bioflavonoids during 4 h. After drying, the bagasse was mixed with boiling distilled water (1 L) in a conical flask for 1 h to remove free soluble sugars, water-soluble pectin and proteins. Then, a boiling solution of HCl (0.05 mol L−1) was used to remove acid-soluble pectin under stirring for 1 h. After filtration and washing with hot water, the residue was submitted to removal of alkali-soluble pectin with boiling solution of NaOH 0.3% (w/v) for 1 h. Finally, soda lignin extraction was carried out based on the procedure reported by Mousavion and Doherty (2010) using a boiling solution of NaOH 3% (w/v) under stirring for 1 h. Using vacuum filtration, a reddish-brown liquid was obtained in the filtrate. After cooling, a solution of H2SO4 (10%) was slowly added to the liquor up to pH 5.5. The solution was left under stirring for another 10 min, followed by further addition of H2SO4 (10%), reaching pH 3.0. At this point, clear signs of precipitation were observed. The mixture was left at 0 °C for 16 h for complete precipitation. Lignin was obtained after vacuum filtration and was washed with small portions of water, acetone and propyl alcohol. Recrystallization was then performed to increase soda lignin purity. The brown solid was dissolved in a NaOH solution (3%, w/v), followed by the aforementioned precipitation procedure using H2SO4 (10%, w/v). The soda lignin was vacuum-filtrated and washed with small portions of water, acetone and propyl alcohol. The solid was freeze-dried for complete removal of water.

2.2 Lignin characterization

Infrared (FTIR) analysis was carried out in an Agilent CARY 630 FTIR spectrophotometer, with 64 scans from 4000 to 400 cm−1, and resolution of 4 cm−1.

Nuclear magnetic resonance (NMR) spectra were obtained in a Bruker Avance 600 MHz spectrometer equipped with a 5-mm probe (TBI) at 25 °C. Isolated soda lignin was dissolved in DMSO-d6 (39.5 ppm) as solvent, being left in sonication bath for 30 min for better dissolution and followed by simple filtration. Two-dimensional HSQC spectrum was obtained at the same equipment with standard TBI probe, 256 scans for f2 and 4 k of f1, with time delay of 2 s.

2.3 Synthesis of silver nanoparticles

A solution of AgNO3 1 mmol L−1 was slowly added under stirring to a soda lignin solution (0.15%) in NaOH (0.1 mol L−1) at room temperature. The reaction was preserved from the sunlight and left at rest.

2.4 Silver nanoparticles characterization

The AgNPs suspension was washed to elevate the pH to neutral. This was done by centrifuging the sample at 14,000 rpm for 30 min at 25 °C, removing the supernatant and then re-dispersing the nanoparticles in ultrapure water.

UV–Vis spectroscopy was carried out in a HP8453 spectrophotometer in the range between 200 and 1000 nm using a 1-cm path length quartz cuvette. Dynamic light scattering (DLS) and zeta potential measurements were performed in a Zetasizer Nano ZS (Malvern Instruments) using a DTS1070 cuvette. Measurements were taken in triplicate, with 15 runs. Transmission electron microscopy images were acquired in a Libra 120 (Carl Zeiss) using 80 kV. The sample was diluted (1:3, v/v) with deionized water before being deposited in the sample holder. Infrared spectrum was acquired in an Agilent CARY 630 FTIR spectrophotometer, with 64 scans from 4000 to 400 cm−1 in resolution of 4 cm−1, using a freeze-dried sample.

3 Results and discussion

After sequential extraction and lyophilization, the orange bagasse soda lignin was isolated in 0.34% yield. This low yield was somewhat expected having seen that the bagasse material is usually poor in lignin (up to 3%). Figure 2 displays 1D and 2D NMR spectra of soda lignin, with the most relevant signal assignments.
Fig. 2

a 1 H NMR spectrum of soda lignin extracted from orange bagasse obtained using water suppression by Gradient-Tailored Excitation (WATERGATE). Structures, abbreviations of monomers and their assignments are shown in color codes; b and c HSQC contour maps of soda lignin extracted from the orange bagasse

A rich region between 6.80 and 8.00 ppm (Fig. 2, in the expanded spectrum) contains peaks that correspond to the aromatic hydrogen atoms of the lignin building blocks. The existence of an abundant aliphatic portion in lignin is seen in the region between 0.00 and 2.00 ppm, while the region between 3.00 and 5.50 ppm has signals attributed to residual sugars and specific dimer structures. Finally, the most shifted region, between 8.00 and 12.00 ppm, contains peaks related to phenolic hydrogen atoms (Li and Lundquist 1994) and/or end aldehyde and carboxylic acid groups.

Due to the specific and complex lignin structure, HSQC spectra are often used for structure assessments and quantification experiments (Mansfield et al. 2012; Wen et al. 2013; Menezes et al. 2016). In Fig. 2b, c, the signals of δC/δH at 55.52/3.75, 62.87/3.71 and 115.31/6.61 correspond to the guaiacyl unit, whereas the 114.35/6.92, 129.06/7.66 and 129.90/7.62 are signals from aromatic structure of p-hydroxyphenyl. Signals of syringyl monomers are found at δC/δH 130.31/6.74 and 130.40/6.99. Also, signals δC/δH at 126.60/7.15 and 128.44/7.21 are derived from the p-hydroxyphenyl, and δC/δH at 62.96/3.28; 3.35 and 100.99/4.53 from the γ-pino/resinol, which are very abundant in lignin structures from various plants (Zeng et al. 2013). Lactone spirodienone produced signals at δCH 100.92/4.22 and 101.42/4.26. Ferulate units are also present in the lignin structure, and their signals are located at 119.51/7.69 ppm. Despite the sequential isolation of the lignin, characteristic peaks such as for xylan at δC/δH 72.12/3.16, 72.59/3.30, 73.86/3.23 and 76.73/3.06, were observed. Terminal –OH groups have assignments at δC/δH 69.67/3.50 and 121.65/5.33. The more intense signals corresponding to p-hydroxyphenyl are in line with what is expected from soda lignin; this lignin type is the one with the higher ratio of activated sites (in C3 and C5) per C9 structure (El Mansouri and Salvadó 2006), which means that positions C3 and C5 in the monolignols are free of methoxy groups.

Silver nanoparticles were produced a few hours after adding silver nitrate solution into the lignin dissolved in alkali medium (0.15%), as denoted by the appearance of a surface plasmon resonance band at 412 nm (Fig. 3). AgNPs were found to be stable for several months (monitored during 6 months), with no signs of aggregation. The strong stabilization of the AgNPs arises from both steric stabilization provided by the biopolymer and from the rather high zeta potential, of − 28.5 ± 3.2 mV. This elevated negative value is most probably due to the reminiscent deprotonated phenolic groups of the biopolymer after the reduction process.
Fig. 3

UV–Vis spectrum of AgNPs suspension produced upon reduction of silver (I) with soda lignin (0.15%) extracted from the orange bagasse. In detail, photograph of the AgNPs suspension displaying the typical yellow color of silver nanoparticles in aqueous medium

Transmission electron microscopy revealed a very homogeneous nanosystem containing spherical nanoparticles. Nanoparticles of 19.1 ± 4.7 nm in size are depicted in Fig. 4. This value contrasts with the one found by dynamic light scattering (DLS), of 73 nm in average (90% of the suspension), since this last technique analyzes not only the metallic core of the nanoparticles but also the organic capping and hydrodynamic sphere around the nanoparticles. The size, morphology and stability of the produced nanoparticles are in line with what is already reported for AgNPs synthesis using lignin (Shen et al. 2014; Hu and Hsieh 2015, 2016).
Fig. 4

Transmission electron micrographs of AgNPs suspension produced upon reduction of silver ions with soda lignin (0.15%) extracted from the orange bagasse

X-ray diffraction analysis of the AgNPs freeze-dried suspension (Fig. 5a) revealed the existence of exclusively Ag0 nanoparticles, which is proved by the reflections at 2θ = 38.1°, 44.4°, 64.4° and 77.2°, which correspond to the pattern of face-centered cubic-indexed facets (111), (200), (220) and (311), respectively (Durán et al. 2016a, b). The absence of reflections corresponding to AgCl nanocrystals indicates that reduction of silver ions was indeed carried out in suspension by lignin, most probably due to the existence of several phenolic groups in the biopolymer.
Fig. 5

a X-ray diffraction pattern of freeze-dried AgNPs suspension produced from orange peel soda lignin. b Comparison between infrared absorption spectra of orange peel soda lignin and corresponding AgNPs suspension

The soda lignin (Fig. 5b) infrared spectrum shows typical and expected bands for this material. The wide band over 3283 cm−1 is related to O–H stretching of hydroxyl and phenolic groups; sharp, low-intensity bands at 2919 and 2852 cm−1 are associated with C–H stretching coming mainly from methoxy groups. Vibrations of the C=C type are present in the band at 1630 cm−1, while bands at 1097 and 1049 cm−1 are associated with primary and secondary alcohol (C–O) vibrations, respectively. The AgNPs FTIR spectrum presents a new and intense band at 1426 cm−1, alongside with a clear distortion of the bands related to C–O vibrations, now at 1152 and 1079 cm−1. This distortion and shift could suggest a stabilization of nanoparticles through hydroxyl groups of the biopolymer.

4 Conclusion

Soda lignin was extracted from orange (Citrus sinensis) bagasse in a low 0.34% yield after a sequential extraction. The natural polymer showed characteristic signals for guaiacyl, p-hydroxyphenyl and syringyl moieties as seen in 1D and 2D NMR spectra. Lignin was then used for the green synthesis of silver nanoparticles, acting as reducing and stabilizing agent. The AgNPs are spherical with average diameters of 19.1 ± 4.7 nm and negative potentials of − 28.5 ± 3.2 mV. Characterization by X-ray diffraction revealed the existence of silver exclusively in the elemental state and infrared absorption spectra disclosed characteristic bands related to groups present in the lignin that were somewhat distorted or shifted after formation of AgNPs.

This paper presents an efficient procedure for soda lignin sequential extraction from orange bagasse that could be applied for any lignin-containing bagasse material, as well as the sustainable manner production of another nanomaterial (nanosilver) with a wide range of possible applications.



The authors would like to acknowledge the fundings provided by Fundação de Amparo à Pesquisa do Estado de São Paulo (Fapesp—2015/12534-5) and Conselho Nacional de Desenvolvimento Científico e Tecnológico—CNPq. We also thank Mr. Douglas Soares da Silva for conducting TEM analyses.

Compliance with ethical standards

Conflict of interest

The authors declare that they have no conflict of interest.


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Copyright information

© Joint Center on Global Change and Earth System Science of the University of Maryland and Beijing Normal University and Springer-Verlag GmbH Germany, part of Springer Nature 2018

Authors and Affiliations

  • Caio H. N. Barros
    • 1
  • Danijela Stanisic
    • 1
  • Bruna F. Morais
    • 1
  • Ljubica Tasic
    • 1
  1. 1.Departamento de Química Orgânica, Laboratório de Química Biológica, Instituto de QuímicaUniversidade Estadual de CampinasCampinasBrazil

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